The present invention relates to the prevention and treatment of Lyme disease in mammals and in particular to immunogenic formulations comprising different serological forms of OspC to retard or prevent the development of Lyme disease. The invention also comprises recombinant methods for the preparation of novel antigens.
Lyme disease or Lyme borreliosis are terms used to describe the diverse clinical symptoms associated with tick-borne spirochetal infections caused by Lyme Disease Borrelia. Common manifestations of Lyme disease include disorders affecting the skin [erythema migrans (EM) or acrodermatitis chronica atrophicans (ACA)], nervous system (neuro-borreliosis), and joints (arthritis) but other organs and tissues may become infected and diseased. Lyme disease has a world-wide distribution and is the most prevalent tick-borne disease in both the United States and Europe. The range of clinical symptoms commonly associated with Lyme disease in Europe is broader than that in the United States, with skin and nervous system disorders being common in Europe but rare in the United States, whereas arthritis is more common in the United States than in Europe. The clinical symptoms in North America appear to be a subset of those observed in Europe.
Lyme disease is typically treated with antibiotics. Treatment may be delayed, however, due to the often complex clinical picture and the lack of widely available, reliable diagnostic tests. If the disease is allowed to proceed to a chronic condition, treatment with antibiotics is more difficult and is not always successful. Furthermore the prospect that permanent damage is induced is likely to be increased during the course of a prolonged infection. Accordingly, a vaccine to prevent Lyme disease is desirable.
Two antigens from Lyme disease Borrelia have been described that can protect against infection/disease by this organism as determined in animal models of Lyme disease. These antigens, OspA and OspC (or xe2x80x9cpCxe2x80x9d), therefore are likely candidates for inclusion in any vaccine designed to protect against Lyme disease. See Simon et al., European patent No. 418,827; Fikrig et al., Science 250: 553-56 (1990); Preac-Mursic et al., Infection 20: 342-49 (1992). OspA and OspC share many characteristics. Both are lipoproteins that are exposed at the cell-surface, (Howe et al., Science 227: 645-46 (1985); Bergstrom et al. Mol. Microbiol. 3: 479-486 (1989)), both are plasmid-encoded (Barbour et al., Science 237: 409-11 (1987); Marconi et al., J. Bacteriol. 175: 926-32 (1993)), the genes for these proteins are present in most strains (Barbour et al., J. Infect. Dis. 152: 478-84 (1985); Marconi et al., J. Bacteriol. 175: 926-32 (1993)), and both exist in multiple serologically distinct forms (Wilske et al., (1989)).
The existence of multiple, serologically distinct forms of these antigens is an obstacle to the development of an OspA and/or OspC vaccine to protect against most, if not all, forms of Lyme disease. For instance, it has been demonstrated by Fikrig et al., J. Immun. 148: 2256-60 (1992), that immunization with one serological form of OspA, such as recombinant OspA of strain N40, need not protect against a challenge with a strain expressing a different OspA, for example, strain 25015. Consequently, it is necessary to develop typing schemes to classify and group the different variants of the antigen i.e., OspA and/or OspC) such that the optimal mixture of serologically distinct forms of the antigen(s) that are needed to give broad protection can be determined.
A serotyping system for OspA has been developed using a limited number of monoclonal antibodies as the typing tools and 7 serotypes of OspA have been described using this methodology. Wilske et al., Ann. N.Y. Acad. sci. 539: 126-43 (1988). Restriction fragment length polymorphism (RFLP) analysis of OspA genes from 55 different European and North American strains identified six distinct genogroups. Wallich et al., Infection and Immunity 60: 4856-66 (1992). OspA proteins from North American isolates seem to be reasonably uniform since twelve of fourteen OspA""s belonged to OspA type I and two to OspA type III. By contrast, the OspA""s from European isolates are much more heterogeneous and include representatives of OspA types I (18), II (17), IV (4) and V (1). Construction of a phylogenetic tree based on sequence data for twelve OspA proteins from individual strains of B. burgdorferi supports the findings of the RFLP analysis but sequence information from isolates from two of the six genogroups is still lacking. At present no typing system exists for OspC.
Another consideration when selecting the appropriate antigens for inclusion in a vaccine is whether they are derived from strains that are epidemiologically important for the disease. In the mid-1970""s it was postulated that pathogenic bacteria arise from a limited number of clones of highly related bacteria that in some way have a selective advantage in causing disease. This clonal hypothesis has since been confirmed. See Achtman et al., J. Infect. Dis. 165: 53-68 (1992). Thus, it is highly likely that among the numerous strains of Lyme disease Borrelia found in nature, only a limited number of xe2x80x9cclonesxe2x80x9d exist that are highly adapted to causing mammalian, and in particular human, disease. In developing a vaccine to protect against disease in mammals and hence also in humans, it is of paramount importance to identify disease associated clones so that efforts can be concentrated against them. Thus it is necessary to elucidate the population structure of the species Lyme disease Borrelia and identify disease associated clones.
To date, a number of methods have been used to resolve the population structure of Lyme disease Borrelia, including (A) RFLP analysis of genomic DNA or of specific genes (LeFebvre et al., J. Clin. Micriobiol. 27: 636-39 (1989); Marconi and Garon, J. Bacteriol 174: 241-44 (1992); Postic et al., Res. Micriobiol. 141: 465-75 (1990); Stahlhammar-Carlemalm et al., Zbl. Bak 274: 28-39 (1990); Adam et al., Infect. Immun. 549: 2579-85 (1991); Wallich et al., Infect. Immun. 60: 4856-66 (1992)), (B) DNA-DNA hybridization (LeFebvre et al., J. Clin. Micriobiol. 27: 636-39 (1989); Postic et al., Res. Micriobiol. 141: 465-75 (1990)), (C) analysis of 16S rRNA by hybridization to oligonucleotide probes (Marconi et al., J. Clin. Micriobiol 30: 628-32 (1992) or by sequencing (Adam et al., Infect. Immun. 59: 2579-85 (1991); Marconi and Garon, J. Bacteriol. 174: 241-44 (1992)), (D) fingerprinting by an arbitrarily primed polymerase chain reaction (Welsh et al., Int. J. System. Bacteriol. 42: 370-77 (1992)), (E) multi-locus enzyme electrophoresis (Boerlin et al., Infect. Immun. 60: 1677-83 (1992)) and (F) serotyping of isolates (Peter and Bretz, Zbl. Baktk. 277: 28-33 (1992)).
There is broad agreement between the results obtained by these different procedures. In general, it appears that Lyme disease Borrelia isolates can be divided into at least three major groups. Indeed, some investigators believe that the genetic distances between members of these groups is sufficient to merit differentiating them into three separate species: B.burgdorferi sensu stricto (type strain B31), B.garinii sp. nov. (type strain 20047) and a species designated B.afzelii or the xe2x80x9cgroup VS461 Borrelia.xe2x80x9d See Baranton et al., Int. J. Syst. Bacteriol. 42: 378-383, 1992; Marconi and Garon, supra.
The significance of the existence of these different groups for vaccine development remains to be fully elucidated. It is clear from the data of Wallich et al., Infection and Immunity 60: 4856-66 (1992), that there is a strong association between the genogroup to which an isolate belongs and the type of OspA that is produced: isolates from the group containing strain B31 (genogroup AAA or B.burgdorferi sensu stricto) produce a type I OspA (all of thirty strains analyzed), isolates from the group containing strain 20047 (genogroup BBB or B.garinii sp. nov.) usually produce a type II (17/19) OspA but types V (1/19) and VI (3/39) were also noted, isolates from the clone containing strain BO23 (genogroup BBA or group VS 461) produce a type IV OspA (4/4), the remaining two isolates (genogroup B, B/A, A) produce a type III OspA.
Lyme disease isolates from North America predominantly belong to one group (genogroup AAA or B.burgdorferi sensu stricto), represented by strain B31, and consequently produce a type I OspA. This suggests that a vaccine containing a type I OspA may be sufficient to protect against most isolates causing Lyme disease in North America at the present time. In Europe the picture is more complex, since all three major clones are found and there is correspondingly an increased diversity in the types of OspA present (genotypes I, II, IV, V, VI). Furthermore, OspA was found not to protect in two studies, conducted using Lyme disease isolates from Europe, which also demonstrated the utility of OspC as a protective antigen. See U.S. patent application Ser. No. 07/903,580; Preac-Mursic et al., Infection 20: 342-49 (1992).
It was not known heretofore whether OspC was clonally inherited, with specific types of OspC restricted to particular groups of Lyme disease isolates (that is, to B.burgdorferi sensu stricto, B.garinii sp. nov. or group VS461). As OspC is plasmid encoded, Marconi et al., J. Bacteriol. 175: 926-32 (1993), it was conceivable that there had been plasmid-mediated transfer of the OspC gene between the different species of Lyme disease isolates. If this were the case, then the different types of OspC which are known to exist but which have not been defined, would not necessarily be clonally inherited.
It is the object of the present invention to provide an effective OspC vaccine, with broad cross protective levels in relation to all strains, against Lyme disease in mammals, by selecting OspC formulations based on defined OspC families resolved by phenotypic typing (the OspC xe2x80x9cserovarxe2x80x9d typing) and RFLP typing analyses, and sequence analysis of a large variety of strains of worldwide origin.
Furthermore, the OspC gene has been discovered to be clonally inherited. Consequently, it now is possible to interpret the results of the OspC typing schemes in view of epidemiological information from a xe2x80x9cCommon Membrane Antigen Typingxe2x80x9d (CMAT) scheme, described below, which can be used to elucidate the clonal population structure of Lyme disease Borrelia strains. By the same token, it also now is possible to select the most appropriate OspC vaccine formulations either (a) to enable one to design vaccines to protect against strains prevalent within defined geographical regions or (b) to protect specifically and preferentially against epidemiological important disease associated clones or clonal clusters of B. burgdorferi. 
In accomplishing these and other objectives, there has been provided, in accordance with one aspect of the present invention, an immunogenic composition comprising
(a) an amount of material comprising (i) one or more OspC antigens of Lyme disease Borrelia substantially purified from each of the 20 currently recognized OspC families of FIG. 11 or (ii) OspC variants or OspC mimetics of said OspC antigens, said OspC variants or OspC mimetics having a structure that is sufficiently similar to native OspC to induce the production of protective antibodies; and
(b) a physiologically-acceptable excipient therefore, wherein said amount is sufficient to elicit, in a mammal susceptible to Lyme borreliosis, an immune response that is protective against Lyme borreliosis.
According to a further embodiment, the above immunogenic composition comprises one or more, preferably two or more, OspC antigens of Lyme disease Borrelia of the 20 currently recognized OspC-families of FIG. 11 or OspC variants or OspC mimetics of said antigens as defined above under (ii), and a physiologically-acceptable excipient as defined above under (b).
In accordance with another aspect of the present invention, an immunogenic composition is provided that comprises an amount of material comprising (i) one or more OspC antigens of Lyme disease Borrelia substantially purified from each of the OspC-families of FIG. 19 expressed by the human disease associated (HDA) clones and clonal clusters or (ii) OspC variants or OspC mimetics of the OspC antigens, said OspC variants or OspC mimetics having a structure that is sufficiently similar to native OspC to induce the production of protective antibodies; and
(b) a physiologically-acceptable excipient therefore, wherein said amount is sufficient to elicit, in a mammal susceptible to Lyme borreliosis, an immune response that is protective against Lyme borreliosis.
As a preferred embodiment, the above immunogenic composition comprises one or more, preferably two or more, OspC antigens of the ospC-families of FIG. 19, or OspC variants or OspC mimetics as defined above under (ii), and a physiologically-acceptable excipient as defined above under (b).
In a preferred embodiment, an immunogenic composition within the present invention is designed to protect against Lyme disease Borrelia strains prevalent within a particular geographic region, such as North America, Europe or Austria. As a preferred embodiment a combined OspA/OspC vaccine, which is superior to a vaccine formulated with either antigen alone is claimed.
The invention also comprises recombinant methods for the production of novel OspC antigens together with DNA sequences, expression vectors, and transformed host cells.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.